Process for the preparation of microspheres containing biologically active components

ABSTRACT

The present invention is directed to a microsphere for the controlled release of a biologically active molecule which comprises a biologically active molecule and an ester of hyaluronic acid or mixtures of said esters of hyaluronic acids, and wherein said biologically active molecule is surrounded by or adhered to said ester of hyaluronic acid, and wherein said microsphere has a diameter of between 1 μm to 100 μm.

This application is a divisional of Ser. No. 08/169,558 filed on Dec.20, 1993, which is a continuation of Ser. No. 07/890,108 filed on May29, 1992, abandoned.

FIELD OF THE INVENTION

An object of the, invention is a composition of microspheres and aprocess for their preparation starting with biodegradable andbioabsorbable semisynthetic polymers and single molecules or mixtures ofthe same having pharmacological activity, allowing the chemicalcharacteristics of the semisynthetic polymer used and the biological orpharmacological activity of the molecules to remain unaltered,guaranteeing controlled release depending on the hydrophilic propertiesof the chemical substituents present on the semisynthetic polymer.

DESCRIPTION OF RELATED ART

Proteins and peptides are today considered to be an important base fortherapeutic agents. Recombinant DNA technology has made it possibile toproduce many such macromolecular agents with interesting and usefulbiological and pharmacological properties. The enormous technologicaladvancement in chemical synthesis has made many polypeptides with highpharmacological activity accessible. For these drugs however problemsstill persist regarding stability and their administration to man. Whenan active principle is administered to man, it is essential to guaranteethat its pharmacological activity will remain unaltered and that itsrelease will be controlled, in order to avoid undesirable side-effects.It is known that these macromolecular agents are not usually efficaciousafter oral administration, since they are rapidly degraded anddeactivated by the proteolytic enzymes present in the gastrointestinaltract.

Even when these macromolecules do resist enzymatic digestion, theirabsorption is often very slight because of their large size. Otherroutes of administration, such as by nose, mouth, vagina, rectum andthrough the skin, have been used for the absorption of proteins andpeptides but bioavailability proved to be low and variable on account ofthe intrinsic characteristics of the active principle. Consequently,these molecules are normally administered by the parenteral route, eventhough this route, too, has its disadvantages, which are mainly linkedto rapid elimination from the bloodstream. Over the past decaderemarkable progress has been made in pharmaceutical technology dedicatedto the preparation of formulations which allow, on the one hand, for theintrinsic activity of proteins to be preserved and, on the other, fortheir controlled release (Langer R., Science 1527, 1990).

The use of synthetic or partially natural polymeric matrixes means thatdrug release is now reproducible, guaranteeing constant concentrationsin the bloodstream, thereby avoiding repeated administrations with theconsequent risk of side effects.

However, the use of these polymeric matrices has given rise to a seriesof new problems linked essentially to the very nature of the polymersused, such as their toxicity and the toxicity of their degradationproducts, biocompatibility, and the removal of deposits of undegradabledebris.

Current research is aimed at identifying and developing bioabsorbableand biodegradable polymers, partly or wholly made of natural substances,capable of the controlled release of biologically and pharmacologicallyactive molecules, which are able to protect these molecules fromdegradation while allowing for their prolonged release, and withoutaffinity for fibrous organic tissues which might alter their releaseproperties, they should not present undesirable reactivity towardspharmacologically active molecules, and neither they, nor theirdegradation products should be immunogenic.

Examples of natural polymers widely used as release systems forpharmacologically active molecules such as hyaluronic acid have beendescribed in U.S. Pat. No. 4,851,521 and U.S. Pat. No. 4,965,353;alginic acid in EP Publ. No. 0251905; chitosan in EP Publ. No. 0341745;and gellan of the acidic polysaccharides.

Hyaluronic acid is a polysaccharide widely distributed in animalorganisms, and is composed of alternating units of D-glucuronic acid andN-acetyl-D-glucosamine. The mean molecular weight varies between 2×104and 7×106 according to the purification method used. Hyaluronic acid hasbeen used, as described, for example, in U.S. Pat. No. 4,772,419, toprevent adhesion and tissue enlargement. U.S. Pat. No. 4,636,524 alsodescribes a release system for biologically active substances to bedispersed in the molecular "cage" formed by the meshwork of hyaluronicacid gel. Hyaluronic acid has also been described in the literature as acarrier for drugs trapped in the biodegradable, collagen-based matrix.In U.S. Pat. Nos. 4,851,521 and 4,965,353, a chemical method is reportedfor the esterification of carboxy groups of hyaluronic acid withtherapeutically active or inactive alcohols (HYAFF). With this chemicalmodification, the chemico-physical properties of the polymer change too,such as the hydrophobic and hydrophilic properties of the polymer, whileits chemical structure of the polysaccharide remains unaltered, as setforth below. ##STR1##

Moreover, various methods for the preparation of systems for thecontrolled release of biologically active substances are described inthe literature.

For instance microspheres containing biologically and/orpharmacologically active molecules are described in the PCT patent WO89/03207. These release systems include the association ofpharmacologically and/or biologically active molecules, denaturable likepolypeptides, with natural polymers (starch, gelatin or albumin). Theuse of hyaluronic and and/or its derivatives as a polymeric carrier toprepare formulations (microspheres) to be used for the release of activesubstances through the mucosa, such as the vaginal or nasal mucosa, isnot described.

The in vitro characterization of the release of pharmacologically activesubstances, not of a proteic or glycosphingolipid nature, frommicrospheres prepared with hyaluronic acid esters is described in thepaper "Microspheres of Hyaluronic Acid Esters Fabrication Methods and invivo Hydrocortisone Release" by Benedetti et al., Journal of ControlledRelease 13, 33-41 (1990).

Of the various technologies developed for the manufacture ofmicrospheres, the most successful have been the "evaporation" and"extraction" techniques. Both of these processes require the preparationof an emulsion of two unmixable liquids. The emulsifying phase, known asthe discontinuous phase, is constituted by microdroplets of a solventcontaining modified hyaluronic acid and the substance, or suitablemixtures of biologically and/or pharmacologically active substances. Theother phase of the emulsion, known as the continuous phase, isrepresented by a second solvent in which the microdroplets arehomogeneously dispersed. When the emulsion is stable, the discontinuousphase is removed either by evaporation or extraction according to thetype of technique employed. It is possible to obtain release systemswith different characteristics according to how the biologically activesubstance or mixture of substances are incorporated in the microspheres.For example, when the active principle is physically dispersed in thepolymer matrix constituting the microspheres, its release is controlledby the diffusion rate of the biologically and/or pharmacologicallyactive substance through the polymer network.

The paper by Benedetti et al. (1990) refers, in particular, to thepossibility of obtaining microspheres by evaporation, because theextraction method produces microspheres with very porous surfaces; and,consequently, the polymeric matrix of which they are constituted has nocontrol over the release of the active principle (corticosteroid). Thepresent invention, surprisingly, provides the possibility of producing,by extraction, smooth-surfaced microspheres which therefore have morecontrol over the release of the substances which are incorporatedtherein.

Another advantage of the extraction technique of the present inventionlies in the possibility of obtaining microspheres with a notably smallerdiameter than those cited in Benedetti et al.

It therefore follows that the use of said microspheres, unlike thosewith a larger diameter, guarantees a greater total surface area andtherefore more contact with the tissues to be treated.

The present invention describes the preparation of microspherescontaining molecules of a proteic nature (such as calcitonin, insulin,immunoglobin, trophic factors such as hCNTF/hNGF) and/or of aglycosphingolipid nature (natural gangliosides or their chemicalderivatives), that is, the preparation of compounds which are quitedifferent in structure, chemical-physical characteristics and stabilityfrom the compound (corticosteroid) discussed in the paper by Benedettiet al. It has been demonstrated, surprisingly, that by using thisrelease system, the proteins associated with the polymer do not undergodegradation and maintain their biological activity.

The present invention also demonstrates the incorporation ofhigh-molecular-weight molecules, i.e., the molecular weight of theincorporated molecules is considerably higher than that of thecorticosteroid.

The possibility of preparing microspheres from HYAFF derivatives withdistinct chemical-physical (hydrophilic/hydrophobic) characteristics,chosen according to the chemical-physical characteristics of thebiologically active molecule used, and where the biologically activemolecule is to be applied, the release time and consequent action of thepharmacologically active molecule, is described according to the presentinvention. As a result of the present invention, it is possible toprepare microspheres from suitable mixtures of HYAFF polymer andpharmacologically and biologically active substances, specificallydesigned according to the type of administration, the type of activesubstance and the desired action of time. It is also possible to preparemicrospheres where the pharmacologically and/or biologically activesubstance or mixture of substances is superficially adsorbed. Theprinciples described above for microspheres and the possibility of apharmacological interaction aimed at the site of action are valid inthis case too.

SUMMARY OF THE INVENTION

Accordingly, to the present invention is directed to a process ofobtaining compositions of microspheres starting from chemically modifiedhyaluronic acid, having different molecular weights, and from solutionsof single agents and suitable mixtures thereof, which exercise abiological and/or pharmacological action, and which can be preparedaccording to the desired site of their desired time of release, the typeof biologically and/or pharmacologically active agent to be released,while leaving intact, the biological and pharmacological properties ofthe agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the levels of calcium in plasma (%) after vaginaladministration of calcitonin in solution.

FIG. 2 shows the levels of calcium in plasma (%) after vaginaladministration of calcitonin associated with mircospheres of HYAFF-11and HYAFF-11 p75.

FIG. 3 shows the effect of different doses of HYAFF-11 microspherescontaining insulin on plasma glucose decrease after nasal administrationin sheep.

FIG. 4 shows the levels of insulin in plasma after nasal administrationto sheep of different doses of HYAFF-11 microspheres.

FIG. 5 shows the plasma levels of GM₁ after intramuscular administrationto rabbits, on its own and with microspheres of different diameters.

FIG. 6 shows the NGF released from microspheres of HYAFF-11 p75.

FIG. 7 shows the NGF released from microspheres of HYAFF-11.

FIG. 8 shows the NGF released from microspheres of HYAFF-7.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The microspheres according to the present invention must have a particlesize of between 1 and 100 μm, preferably between 1 and 15 μm, and theymust have smooth surfaces. The following examples are purelyillustrative of how to obtain the microspheres according to the presentinvention and their use and shall not be construed as limiting the scopeof the invention. The molecular weight of the hyaluronic acid esterderivatives (HYAFF) which makes up the microspheres of the presentinvention can be, for example, in the range of about 100,000-2,000,000Daltons, with a preferred range being between about 100,000-200,000Daltons or between about 500,000-700,000 Daltons.

EXAMPLE 1

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353) is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying between 5 and 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide such as human insulin, at a predetermined concentration, forexample 5 I.U. per mg of polymer, is added to the solution. The mixtureobtained will be referred to hereinafter as the discontinuous phase. Atthe same time, a mixture is prepared in a suitable reactor ofhigh-viscosity mineral oil containing Arlacel^(R), a non-ionicsurface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase.

The continuous phase is kept at 25° C. while being stirred at a fixedspeed of 1000 RPM, then the discontinuous phase, prepared as previouslydescribed, is added to it. In these conditions, emulsification of thetwo phases is instantaneous. The ratio between the two phases(discontinuous and continuous) is about 1 to 16.

After 15 minutes of stirring, acetylacetate is added.

This solvent mixes perfectly with the two phases of the emulsion but itis a nonsolvent for the polymer and the human insulin polypeptide. Ithas been proven that the volume of extracting solvent needed to obtaincomplete extraction is two and a half times the total volume of theemulsion. To facilitate extraction the stirring speed is set at1400-1500 RPM for 10 minutes and then lowered to 500 RPM. The suspensionthus obtained continues to be stirred while being pumped with a screwpump through a filter press set at 1 atmosphere. Once this filtration iscomplete, it is pumped through a filter of normal-hexane, this being asolvent with the double action of "drying" the preparation andsolubilizing any residue surfactant which may be present on the surfaceof the microspheres. The product is then put in suitable containers andstored at 4° C.

In these working conditions the resulting mean particle size is 10 μm.

The quantity of incorporated insulin is 4 IU per mg of microspheres.

EXAMPLE 2

A hyaluronic acid ester wherein all the carboxy groups of hyaluronicacid are esterified with benzyl alcohol (HYAFF-11, as described in theU.S. Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide at a concentration varying from 5 to 10%weight/volume, generally 7% p/v. The solution obtained will be referredto hereinafter as the discontinuous phase. At the same time, a mixtureis prepared in a suitable reactor of high-viscosity mineral oilcontaining Arlacel^(R), a non-ionic surface-active agent, at aconcentration of 1% p/v.

This mixture will be referred to hereinafter as the continuous phase.The continuous phase is kept at a temperature of 25° C. and stirred at arate of 1000 RPM, while the discontinuous phase, prepared as previouslydescribed, is added to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 RPM for 10 minutes, then loweredto 500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and solubilizing any residue surfactant which may bepresent on the surface of the microspheres. The product is then put insuitable containers and stored at 4° C. The microspheres thus preparedare suspended in a phosphate buffer solution (0.01M) (ionicstrength=0.15M), containing a concentration of insulin such that aprotein titer of 2 U.I per mg of suspended microspheres is reached.After 15 minutes stirring with a semiautomatic system the suspension isimmersed in liquid nitrogen until it is completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 15 μm. The quantity of incorporated insulin is2 IU per mg of microsferes.

EXAMPLE 3

A hyaluronic acid ester wherein 75% of the carboxy groups of hyaluronicacid are esterified with benzyl alcohol while the remaining part issalified with sodium (HYAFF-11 p75, as described in the U.S. Pat. No.4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide such as human insulin, at a predetermined concentration, forexample 5 I.U. per mg of polymer, is added to the solution. The mixturethus obtained will be referred to hereinafter as the discontinuousphase. At the same time a mixture is prepared in a suitable reactor ofhigh-viscosity mineral oil containing Arlacel^(R), a non-ionicsurface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase.The continuous phase is kept at a temperature of 25° C. and stirred at arate of 1000 RPM, while the discontinuous phase, prepared as previouslydescribed, is added to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the human insulin polypeptide. Ithas been proven that the volume of extracting solvent needed to obtaincomplete extraction is two and a half times the total volume of theemulsion. To facilitate extraction, the stirring speed is set at1400-1500 RPM for 10 minutes, then lowered to 500 RPM. The suspensionthus obtained continues to be stirred, while being pumped by a screwpump through a filter press set at 1 atmosphere. Once this filtration iscomplete, it is pumped through a filter of normal-hexane, this being asolvent with the double action of "drying" the preparation andsolubilizing any residue surfactant which may be present on the surfaceof the microspheres. The product is then put in suitable containers andstored at 4° C.

The mean particle size is 20 μm.

The quantity of incorporated insulin is 4 IU per mg of microspheres.

EXAMPLE 4

A hyaluronic acid ester wherein all the carboxy groups of hyaluronicacid are esterified with ethyl alcohol (HYAFF-7, as described in theU.S. Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, such as human insulin, at a predetermined concentration,for example 5 I.U. per mg of polymer, is added to the solution. Themixture thus obtained will be referred to hereinafter as thediscontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase.The continuous phase is kept at temperature of 25° C. and stirred at arate of 1000 RPM, while the discontinuous phase, prepared as previouslydescribed, is added to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the human insulin polypeptide. Ithas been proven that the volume of extracting solvent needed to obtaincomplete extraction is two and a half times the total volume of theemulsion. To facilitate extraction, the stirring speed is set at1400-1500 RPM for 10 minutes, then lowered to 500 RPM. The suspensionthus obtained is stirred while being pumped by a screw pump, through afilter press set at 1 atmosphere. Once this filtration is complete, itis pumped through a filter of normal-hexane, this being a solvent withthe double action of "drying" the preparation and solubilizing anyresidue surfactant which may be present on the surface of themicrospheres. The product is then put in suitable containers and storedat 4° C.

The dimensions of the microshperes and the mean particle size is 30 μm.

The quantity of incorporated insulin is 4 I.U. per mg of microspheres.

EXAMPLE 5

A hyaluronic acid ester wherein all the carboxy groups of hyaluronicacid are esterified with ethyl alcohol (HYAFF-7, as described in theU.S. Pat. No. 4,965,353) is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. The solution obtained will be referredto hereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable container of high-viscosity mineral oilcontaining Arlacel^(R), a non-ionic surface-active agent, at aconcentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 for 10 minutes, then lowered to500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once filtration is complete, it is pumped through a normal-hexanefilter, this being a solvent with the double action of "drying" thepreparation and solubilizing any residue surfactant which may be presenton the surface of the microspheres. The product is then put in suitablecontainers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength=0.15M), containing a concentration ofinsulin such that a protein titer of 2 I.U. per mg of suspendedmicrospheres is reached. After 15 minutes of stirring with asemiautomatic system the container is immersed in liquid nitrogen untilthe suspension has completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 30 μm.

The quantity of incorporated insulin is 2 I.U. per mg of microspheres.

EXAMPLE 6

A hyaluronic acid ester wherein all the carboxy groups of hyaluronicacid are esterified with benzyl alcohol (HYAFF-11, as described in theU.S. Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example Nerve Growth Factor (NGF), at a predeterminedconcentration, for example 0.01% of the weight of the polymer mass, isadded to the solution. The mixture thus obtained will be referred tohereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it. In these conditions, emulsification of the two phases isinstantaneous. The ratio between the two phases (discontinuous andcontinuous) is about 1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide NGF. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

In these working conditions the resulting mean particle size is 10 μm.

The quantity of incorporated NGF is 10 ng per mg of microspheres.

EXAMPLE 7

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example Nerve Growth Factor (NGF), at a predeterminedconcentration, for example 0.01% of the weight of the polymer mass, isadded to the solution. The solution obtained will be referred tohereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 for 10 minutes, then lowered to500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and solubilizing any residue surfactant which may bepresent on the surface of the microspheres. Th e product is then put insuitable containers and stored at 4° C. The microspheres thus preparedare suspended in a phosphate buffer solution (0.01M) (ionicstrength=0.15M), containing a concentration of NGF such that a proteintiter equal to 0.01% by weight of the suspended microspheres is reached.After 15 minutes of stirring with a semiautomatic system the containeris immersed in liquid nitrogen until the suspension is completelyfrozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 10 μm.

The quantity of incorporated NGF is 10 ng per mg of microspheres.

EXAMPLE 8

A hyaluronic acid ester where 75% of the carboxy groups of hyaluronicacid are esterified with benzyl alcohol while the remaining part issalified with sodium (HYAFF-11 p75, as described in the U.S. Pat. No.4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. once the polymer has solubilized, apolypeptide, for example NGF, at a predetermined concentration, forexample 0.01% of the weight of the polymer mass, is added to thesolution. The mixture thus obtained will be referred to hereinafter asthe discontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the NGF polypeptide. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any possibleresidues of surfactant which may be present on the surface of themicrospheres. The product is then put in suitable containers and storedat 4° C.

The mean particle size is 15 μm.

The quantity of incorporated NGF is 10 ng per mg of microspheres.

EXAMPLE 9

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example NGF, at a predetermined concentration, forexample 0.01% of the weight of the polymer mass, is added to thesolution. The mixture thus obtained will be referred to hereinafter asthe discontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or for the NGF polypeptide. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

The mean particle size is 30 μm.

The quantity of incorporated NGF is 10 ng per mg of microspheres.

EXAMPLE 10

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. The solution obtained will be referredto hereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it. In these conditions, emulsification of the two phases isinstantaneous. The ratio between the two phases (discontinuous andcontinuous) is about 1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 for 10 minutes, then lowered to500 RPM. The suspension thus obtained is stirred while being pumped byscrew pump through a filter press set at 1 atmosphere. Once thisfiltration is complete, it is pumped through a filter of normal-hexane,this being a solvent with the double action of "drying" the preparationand solubilizing any residue surfactant which may be present on thesurface of the microspheres. The product is then put in suitablecontainers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength=0.15M), containing a concentration ofNGF such that a protein titer equal to 0.01% in weight of the suspendedmicrospheres is reached. After 15 minutes of stirring with asemiautomatic system the container is immersed in liquid nitrogen untilthe suspension is completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The dimensions of the microspheres and the mean particle size is 30 μm.

The quantity of incorporated NGF is 10 ng per mg of microspheres.

EXAMPLE 11

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example CNTF (Ciliary Neuronotrofic Factor), at apredetermined concentration, for example 0.01% of the weight of thepolymer mass, is added to the solution.

The mixture thus obtained will be referred to hereinafter as thediscontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide CNTF. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

In these working conditions the resulting mean particle size is 10 μm.

The quantity of incorporated CNTF is 10 ng per mg of microspheres.

EXAMPLE 12

A hyaluronic acid ester, where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. The solution obtained will be referredto hereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 for 10 minutes, then lowered to500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and solubilizing any residue surfactant which may bepresent on the surface of the microspheres. The product is then put insuitable containers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength=0.15M), containing a concentration ofCNTF (Ciliary Neuronotrofic Factor) such that a protein titer equal to0.01% in weight of the suspended microspheres is reached. After 15minutes of stirring with a semiautomatic system the container isimmersed in liquid nitrogen until the suspension is completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 10 μm.

The quantity of incorporated CNTF is 10 ng per mg of microspheres.

EXAMPLE 13

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example CNTF (Ciliary Neronotrophic Factor), at thepredetermined concentration, for example 0.01% of the weight of thepolymer mass, is added to the solution.

This mixture will be referred to hereinafter as the discontinuous phase.At the same time a mixture is prepared in a suitable reactor ofhigh-viscosity mineral oil containing Arlacel^(R), a non-ionicsurface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide CNTF. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

The mean particle size is 30 μm.

The quantity of incorporated CNTF is 10 ng per mg of microspheres.

EXAMPLE 14

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. The solution obtained will be referredto hereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 for 10 minutes, then lowered to500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and of solubilizing any possible residues of surfactantwhich may be present on the surface of the microspheres. The product isthen put in suitable containers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength=0.15M) containing a concentration ofCNTF (Ciliary Neuronotrophic Factor) such that a protein titer equal to0.01% in weight of the suspended microspheres is reached. After 15minutes of stirring with a semiautomatic system the container isimmersed in liquid nitrogen until the suspension is completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 30 μm.

The quantity of incorporated CNTF is 10 ng per mg of microspheres.

EXAMPLE 15

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example NGF at the predetermined concentration, forexample 0.01% of the weight of the polymer mass, and a gangliosidemixture having as major components, GM1 21%, GD12 40%, GD1b 16% and GT1b19% (Cronassial®) are added at a ratio of 1:1000 in terms of weight.

The mixture thus obtained will be referred to hereinafter as thediscontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide NGF or the GAmixture (Cronassial). It has been proven that the volume of extractingsolvent needed to obtain complete extraction is two and a half times thetotal volume of the emulsion. To facilitate extraction, the stirringspeed is set at 1400-1500 RPM for 10 minutes, then lowered to 500 RPM.The suspension thus obtained continues to be stirred, while being pumpedby a screw pump through a filter press set at 1 atmosphere. Once thisfiltration is complete, it is pumped through a filter of normal-hexane,this being a solvent with the double action of "drying" the preparationand solubilizing any residue surfactant which may be present on thesurface of the microspheres. The product is then put in suitablecontainers and stored at 4° C.

In these working conditions, the dimensions of the microspheres and themean particle size is 10 μm.

The quantity of incorporated NGF and GA is 10 ng and 10 μg respectivelyper milligram of microspheres.

EXAMPLE 16

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, aganglioside mixture (Cronassiall) at the predetermined concentration,for example 20% of the weight of the polymer, is added to the solution.The solution obtained will be referred to hereinafter as thediscontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it. In these conditions, emulsification of the two phases isinstantaneous. The ratio between the two phases (discontinuous andcontinuous) is about 1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the ganglioside mixture. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength=0.15M), containing a concentration ofNGF such that a protein titer equal to 0.02% in weight of the suspendedmicrospheres is reached, and such that the weight ratio of 1:1000(NGF:gangliosides) is maintained.

After 15 minutes of stirring with a semiautomatic system the containeris immersed in liquid nitrogen until the suspension is completelyfrozen. Once frozen, the suspension is freeze-dried for 24 hrs and theproduct stored at 4° C.

The mean particle size is 10 μm.

The quantity of incorporated NGF and GA is 20 ng and 20 μg,respectively, per milligram of microspheres.

EXAMPLE 17

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example NGF at the predetermined concentration, forexample 0.01% of the weight of the polymer mass, and a gangliosidemixture (Cronassial®) are added to the solution at a weight ratio of1:1000 (NGF:Cronassial®). The mixture thus obtained will be referred tohereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer, the polipeptide (NGF) or the GAmixture. It has been proven that the volume of extracting solvent neededto obtain complete extraction is two and a half times the total volumeof the emulsion. To facilitate extraction, the stirring speed is set at1400-1500 RPM for 10 minutes, then lowered to 500 RPM. The suspensionthus obtained continues to be stirred, while being pumped by a screwpump through a filter press set at 1 atmosphere. Once this filtration iscomplete, it is pumped through a filter of normal-hexane, this being asolvent with the double action of "drying" the preparation andsolubilizing any residue surfactant which may be present on the surfaceof the microspheres. The product is then put in suitable containers andstored at 4° C.

The dimensions of the microspheres and the mean particle size is 30 μm.

The quantities of incorporated NGF and GA are 10 ng and 10 μgrespectively per milligram of microspheres.

EXAMPLE 18

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, aganglioside mixture (Cronassial®) at the predetermined concentration,for example 20% of the weight of the polymer, is added to the solution.The solution thus obtained will be referred to hereinafter as thediscontinuous phase.

At the same time a mixture is prepared in a suitable reactor ofhigh-viscosity mineral oil containing Arlacel^(R), a non-ionicsurface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the ganglioside mixture. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength=0.15M), containing a concentration ofNGF such that a protein titer equal to 0.02% in weight of the suspendedmicrospheres is reached and such that the weight ratio of 1:1000(NGF:gangliosides) is maintained. After 15 minutes of stirring with asemiautomatic system the container is immersed in liquid nitrogen untilthe suspension is completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 30 μm.

The quantities of incorporated NGF and GA are 20 ng and 20 μgrespectively per milligram of microspheres.

EXAMPLE 19

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with dodecyl alcohol (HYAFF-73, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, tothe solution are added a polypeptide, for example NGF at thepredetermined concentration, for example 0.01% of the weight of thepolymer mass, and a ganglioside mixture (Cronassial®) in a weight ratioof 1:1000 (NGF:Cronassial®). The mixture thus obtained will be referredto hereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v. This mixture will be referred to hereinafter as the continuousphase. It is kept at a temperature of 25° C. and stirred at a rate of1000 RPM, while the discontinuous phase, prepared as previouslydescribed, is added to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide (NGF) or the GAmixture (Cronassial®). It has been proven that the volume of extractingsolvent needed to obtain complete extraction is two and a half times thetotal volume of the emulsion. To facilitate extraction, the stirringspeed is set at 1400-1500 RPM for 10 minutes, then lowered to 500 RPM.The suspension thus obtained continues to be stirred, while being pumpedby a screw pump through a filter press set at 1 atmosphere. Once thisfiltration is complete, it is pumped through a filter of normal-hexane,this being a solvent with the double action of "drying" the preparationand solubilizing any residue surfactant which may be present on thesurface of the microspheres. The product is then put in suitablecontainers and stored at 4° C. In these working conditions the resultingmean particle size is 20 μm.

The quantities of incorporated NGF and GA are 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 20

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, tothe solution are added a polypeptide, for example NGF at a predeterminedconcentration, for example 0.01% of the weight of the polymer mass, anda monosialoganglioside GM1 in a weight ratio of 1:1000 (NGF:GM1).

The mixture thus obtained will be referred to hereinafter as thediscontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polipeptide (NGF) or themonosialoganglioside GM1. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 RPM for 10 minutes, then loweredto 500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, a solvent with the double action of "drying" thepreparation and solubilizing any residue surfactant which may be presenton the surface of the microspheres. The product is then put in suitablecontainers and stored at 4° C.

In these working conditions, the resulting mean particle size is 10 μm.The quantities of NGF and monosialoganglioside GM1 are 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 21

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, tothe solution are added a polypeptide, for example NGF at a predeterminedconcentration, for example 0.01% of the weight of the polymer mass, andmonosialoganglioside GM1 in a ratio of 1:1000 (NGF:GM1). This mixturewill be referred to hereinafter as the discontinuous phase. At the sametime a mixture is prepared in a suitable reactor of high-viscositymineral oil containing Arlacel^(R), a non-ionic surface-active agent, ata concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer, the polypeptide (NGF) or themonosialoganglioside GM1. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 RPM for 10 minutes, then loweredto 500 RPM. The suspension thus obtained is stirred while being pumpedby screw pump through a filter press set at 1 atmosphere. Once thisfiltration is complete, it is pumped through a filter of normal-hexane,this being a solvent with the double action of "drying" the preparationand solubilizing any residue surfactant which may be present on thesurface of the microspheres. The product is then put in suitablecontainers and stored at 4° C.

The mean particle size is 30 μm.

The quantities of NGF and monosialoganglioside GM1 are 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 22

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example CNTF, at a predetermined concentration, forexample 0.01% of the weight of the polymer mass, and a gangliosidemixture (Cronassial®) are added to the solution in a ratio of 1:1000(CNTF:gangliosides). The mixture thus obtained will be referred tohereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide CNTF. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

In these working conditions, the resulting mean particle size is 10 μm.The quantity of incorporated CNTF and GA is 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 23

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, aganglioside mixture at a predetermined concentration, for example 20% ofthe weight of the polymer, is added to the solution.

The solution obtained will be referred to hereinafter as thediscontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the ganglioside mixture. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the total volume of the emulsion. Tofacilitate extraction the stirring rate was set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is pumpedthrough a filter of normal-hexane, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength=0.15M) containing a concentration ofCNTF such that a protein titer equal to 0.02% of the weight of thesuspended microspheres is reached, and such that the weight ratio of1:1000 (CNTF:gangliosides) is maintained. After 15 minutes of stirringwith a semiautomatic system the container is immersed in liquid nitrogenuntil the suspension is completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 10 μm.

The quantities of incorporated CNTF and GA are 20 ng and 20 μg,respectively, per milligram of microspheres.

EXAMPLE 24

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example CNTF at a predetermined concentration, forexample 0.01% of the weight of the polymer mass, and a gangliosidemixture (Cronassial®) in a weight ratio of 1:1000 (CNTF:Cronassial®) areadded to the solution. The mixture thus obtained will be referred tohereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer, the polypeptide (CNTF) or the GAmixture (Cronassial®). It has been proven that the volume of extractingsolvent needed to obtain complete extraction is two and a half times thevolume of the emulsion. To facilitate extraction, the stirring speed isset at 1400-1500 RPM for 10 minutes, then lowered to 500 RPM. Thesuspension thus obtained continues to be stirred, while being pumped bya screw pump through a filter press set at 1 atmosphere. Once thisfiltration is complete, it is pumped through a filter of normal-hexane,this being a solvent with the double action of "drying" the preparationand solubilizing any possible residues of surfactant which may bepresent on the surface of the microspheres. The product is then put insuitable containers and stored at 4° C.

The mean particle size is 30 μm.

The quantities of incorporated CNTF and GA are 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 25

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, aganglioside mixture (Cronassial®) at the predetermined concentration,for example 10% of the weight of the polymer, is added to the solution.The solution obtained will be referred to hereinafter as thediscontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the ganglioside mixture. It has beenproven that the volume of extracting solvent needed to obtain completeextraction is two and a half times the volume of the emulsion. Tofacilitate extraction, the stirring speed is set at 1400-1500 RPM for 10minutes, then lowered to 500 RPM. The suspension thus obtained continuesto be stirred, while being pumped by a screw pump through a filter pressset at 1 atmosphere. Once this filtration is complete, it is passedthrough a normal-hexane filter, this being a solvent with the doubleaction of "drying" the preparation and solubilizing any residuesurfactant which may be present on the surface of the microspheres. Theproduct is then put in suitable containers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength 0.15 M), containing a concentration ofCNTF such that a protein titer equal to 0.01% in weight of the suspendedmicrospheres is reached and such that the weight ratio of 1:1000(CNTF:gangliosides) is maintained. After 15 minutes of stirring with asemiautomatic system the container is immersed in liquid nitrogen untilthe suspension is completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 30 μm.

The quantities of incorporated CNTF and GA are 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 26

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example CNTF, at a predetermined concentration, forexample 0.01% of the weight of the polymer mass, and themonosialoganglioside GM1 (Sygen) are added to the solution in a ratio of1:1000 (CNTF:Sygen). The mixture thus obtained will be referred tohereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide CNTF or themonosialoganglioside GM1 (Sygen). It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 RPM for 10 minutes, then loweredto 500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and solubilizing any residue surfactant which may bepresent on the surface of the microspheres. The product is then put insuitable containers and stored at 4° C.

In these working conditions the resulting mean particle size is 10 μm.The quantity of incorporated CNTF and GM1 is 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 27

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example CNTF, at a predetermined concentration, forexample 0.01% of the weight of the polymer mass, and inner esters of theganglioside mixture (AGF1) are added to the solution in a ratio of1:1000 (CNTF:AGF1). The mixture thus obtained will be referred tohereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide CNTF or the inneresters of the ganglioside mixture (AGF1). It has been proven that thevolume of extracting solvent needed to obtain complete extraction is twoand a half times the total volume of the emulsion. To facilitateextraction, the stirring speed is set at 1400-1500 RPM for 10 minutes,then lowered to 500 RPM. The suspension thus obtained continues to bestirred, while being pumped by a screw pump through a filter press setat 1 atmosphere. Once this filtration is complete, it is pumped througha filter of normal-hexane, this being a solvent with the double actionof "drying" the preparation and solubilizing any residue surfactantwhich may be present on the surface of the microspheres. The product isthen put in suitable containers and stored at 4° C.

In these working conditions the resulting mean particle size is 10 μm.The quantity of incorporated CNTF and AGF1 is 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 28

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example CNTF, at a predetermined concentration, forexample 0.01% of the weight of the polymer mass, and themonosialoganglioside GM1 inner ester (AGF2) are added to the solution ina ratio of 1:1000 (CNTF:AGF2). The mixture thus obtained will bereferred to hereinafter as the discontinuous phase. At the same time amixture is prepared in a suitable reactor of high-viscosity mineral oilcontaining Arlacel^(R), a non-ionic surface-active agent, at aconcentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it. In these conditions, emulsification of the two phases isinstantaneous. The ratio between the two phases (discontinuous andcontinuous) is about 1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide CNTF or themonosialoganglioside GM1 inner ester (AGF2). It has been proven that thevolume of extracting solvent needed to obtain complete extraction is twoand a half times the total volume of the emulsion. To facilitateextraction, the stirring speed is set at 1400-1500 RPM for 10 minutes,then lowered to 500 RPM. The suspension thus obtained continues to bestirred, while being pumped by a screw pump through a filter press setat 1 atmosphere. Once this filtration is complete, it is pumped througha filter of normal-hexane, this being a solvent with the double actionof "drying" the preparation and solubilizing any residue surfactantwhich may be present on the surface of the microspheres. The product isthen put in suitable containers and stored at 4° C.

In these working conditions the resulting mean particle size is 10 μm.The quantity of incorporated CNTF and AGF2 is 10 ng and 10 μg,respectively, per milligram of microspheres.

EXAMPLE 29

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 8% w/v. Once the polymer has solubilized, tothis is added a solution of GM1 at a predetermined concentration, forexample 20% of the weight of the polymer mass. The solution thusobtained will be referred to hereinafter as the discontinuous phase. Atthe same time a mixture is prepared in a suitable reactor ofhigh-viscosity mineral oil containing Arlacel^(R), a non-ionicsurface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 700 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 RPM for 10 minutes, then loweredto 500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and solubilizing any residue surfactant which may bepresent on the surface of the microspheres. The product is then put insuitable containers and stored at 4° C. In these working conditions theresulting mean particle size is 40 μm. The quantity of incorporated GM₁is 180 μg per milligram of microspheres.

EXAMPLE 30

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 8% w/v. Once the polymer has solubilized, tothis is added a solution of GM1 at a predetermined concentration, forexample 20% of the weight of the polymer mass. The mixture thus obtainedwill be referred to hereinafter as the discontinuous phase. At the sametime a mixture is prepared in a suitable reactor of high-viscositymineral oil containing Arlacel^(R), a non-ionic surface-active agent, ata concentration of 1% w/v. This mixture will be referred to hereinafteras the continuous phase. It is kept at a temperature of 25° C. andstirred at a rate of 1000 RPM, while the discontinuous phase, preparedas previously described, is added to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 RPM for 10 minutes, then loweredto 500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and solubilizing any residue surfactant which may bepresent on the surface of the microspheres. The product is then put insuitable containers and stored at 4° C. In these working conditions theresulting mean particle size is 10 μm. The quantity of incorporated GM₁is 180 μg per milligram of microspheres.

EXAMPLE 31

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353) is dissolved in an aprotic solvent such asdimethylsulfoxide at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. The solution obtained will be referredto hereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it. In these conditions, emulsification of the two phases isinstantaneous.

The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction the total volumeof the emulsion, in order to obtain complete extraction. To facilitateextraction, the stirring speed is set at 1400-1500 for 10 minutes, thenlowered to 500 RPM. The suspension thus obtained continues to bestirred, while being pumped by a screw pump through a filter press setat 1 atmosphere. Once this filtration is complete, it is pumped througha filter of normal-hexane, this being a solvent with the double actionof "drying" the preparation and solubilizing any residue surfactantwhich may be present on the surface of the microspheres. The product isthen put in suitable containers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M), containing a concentration of immunoglobulin such thata protein titer of 3 μg per mg of suspended microspheres is reached.After 15 minutes of stirring with a semiautomatic system the containeris immersed in liquid nitrogen until the suspension is completelyfrozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 10 μm.

The quantity of incorporated immunoglobulin is 2.5 μg per mg ofmicrospheres.

EXAMPLE 32

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example calcitonin, dissolved in hydrochloric acid atpH=3, at the predetermined concentration, for example 15 I.U. per mg ofpolymer, is added to the solution. The mixture thus obtained will bereferred to hereinafter as the discontinuous phase. At the same time amixture is prepared in a suitable reactor of high-viscosity mineral oilcontaining Arlacel^(R), a non-ionic surface-active agent, at aconcentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide calcitonin. It hasbeen proven that the volume of extracting solvent needed to obtaincomplete extraction is two and a half times the total volume of theemulsion. To facilitate extraction, the stirring speed is set at1400-1500 RPM for 10 minutes, then lowered to 500 RPM. The suspensionthus obtained continues to be stirred, while being pumped by a screwpump through a filter press set at 1 atmosphere. Once this filtration iscomplete, it is pumped through a filter of normal-hexane, this being asolvent with the double action of "drying" the preparation andsolubilizing any residue surfactant which may be present on the surfaceof the microspheres. The product is then put in suitable containers andstored at 4° C.

In these working conditions the resulting mean particle size is 10 μm.The quantity of incorporated calcitonin is 13 I.U. per mg ofmicrospheres.

EXAMPLE 33

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with benzyl alcohol (HYAFF-11, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide, at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. The solution obtained will be referredto hereinafter as the discontinuous phase. At the same time a mixture isprepared in a suitable reactor of high-viscosity mineral oil containingArlacel^(R), a non-ionic surface-active agent, at a concentration of 1%w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring rate is set at 1400-1500 RPM for 10 minutes, then loweredto 500 RPM. The suspension thus obtained is stirred while being pumpedthrough a screw pump through a filter press set at 1 atmosphere. Oncethis filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and solubilizing any residue surfactant which may bepresent on the surface of the microspheres. The product is then put insuitable containers and stored at 4° C.

The microspheres thus prepared are suspended in a phosphate buffersolution (0.01M) (ionic strength=0.1M) at pH=7, containing aconcentration of calcitonin such that a protein titer of 15 I.U. per mgof suspended microspheres is reached. After 15 minutes of stirring witha semiautomatic system the container is immersed in liquid nitrogenuntil the suspension is completely frozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C. The mean particle size is 10 μm. The quantity ofincorporated calcitonin is 13 IU/mg of microspheres.

EXAMPLE 34

A hyaluronic acid ester where 75% of the carboxy groups of hyaluronicacid are esterified with benzyl alcohol while the remaining part issalified with sodium (HYAFF-11 p75, as described in the U.S. Pat. No.4,965,353), is dissolved in an aprotic solvent such as dimethylsulfoxideat a concentration varying from 5 to 10% weight/volume, generally 7%w/v. Once the polymer has solubilized, a polypeptide, for examplecalcitonin, dissolved in hydrochloric acid at pH=3, at a predeterminedconcentration, for example 15 I.U. per mg of polymer is added to thesolution. The mixture thus obtained will be referred to hereinafter asthe discontinuous phase. At the same time a mixture is prepared in asuitable reactor of high-viscosity mineral oil containing Arlacel^(R), anon-ionic surface-active agent, at a concentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polypeptide calcitonin. It hasbeen proven that the volume of extracting solvent needed to obtaincomplete extraction is two and a half times the total volume of theemulsion. To facilitate extraction, the stirring speed is set at1400-1500 RPM for 10 minutes, then lowered to 500 RPM. The suspensionthus obtained continues to be stirred, while being pumped by a screwpump through a filter press set at 1 atmosphere. Once this filtration iscomplete, it is pumped through a filter of normal-hexane, this being asolvent with the double action of "drying" the preparation andsolubilizing any residue surfactant which may be present on the surfaceof the microspheres. The product is then put in suitable containers andstored at 4° C.

The mean particle size is 15 μm. The quantity of incorporated calcitoninis 13 I.U per mg of microspheres.

EXAMPLE 35

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. Once the polymer has solubilized, apolypeptide, for example calcitonin, dissolved in hydrochloric acid atpH=3, at a predetermined concentration, for example 15 I.U. per mg ofpolymer, is added to the solution. The mixture thus obtained will bereferred to hereinafter as the discontinuous phase. At the same time amixture is prepared in a suitable reactor of high-viscosity mineral oilcontaining Arlacel^(R), a non-ionic surface-active agent, at aconcentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it.

In these conditions, emulsification of the two phases is instantaneous.The ratio between the two phases (discontinuous and continuous) is about1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer or the polipeptide calcitonin. It hasbeen proven that the volume of extracting solvent needed to obtaincomplete extraction is two and a half times the total volume of theemulsion. To facilitate extraction, the stirring speed is set at1400-1500 RPM for 10 minutes, then lowered to 500 RPM. The suspensionthus obtained continues to be stirred, while being pumped by a screwpump through a filter press set at 1 atmosphere. Once this filtration iscomplete, it is pumped through a filter of normal-hexane, this being asolvent with the double action of "drying" the preparation andsolubilizing any residue surfactant which may be present on the surfaceof the microspheres. The product is then put in suitable containers andstored at 4° C.

The mean particle size is 30 μm. The quantity of incorporated calcitoninis 13 I.U. per mg of microspheres.

EXAMPLE 36

A hyaluronic acid ester where all the carboxy groups of hyaluronic acidare esterified with ethyl alcohol (HYAFF-7, as described in the U.S.Pat. No. 4,965,353), is dissolved in an aprotic solvent such asdimethylsulfoxide at a concentration varying from 5 to 10%weight/volume, generally 7% w/v. The solution thus obtained will bereferred to hereinafter as the discontinuous phase. At the same time amixture is prepared in a suitable reactor of high-viscosity mineral oilcontaining Arlacel^(R), a non-ionic surface-active agent, at aconcentration of 1% w/v.

This mixture will be referred to hereinafter as the continuous phase. Itis kept at a temperature of 25° C. and stirred at a rate of 1000 RPM,while the discontinuous phase, prepared as previously described, isadded to it. In these conditions, emulsification of the two phases isinstantaneous. The ratio between the two phases (discontinuous andcontinuous) is about 1 to 16.

After stirring for 15 minutes, ethyl acetate is added.

This solvent can be mixed perfectly with the two emulsion phases, but itis not a solvent for the polymer. It has been proven that the volume ofextracting solvent needed to obtain complete extraction is two and ahalf times the total volume of the emulsion. To facilitate extraction,the stirring speed is set at 1400-1500 for 10 minutes, then lowered to500 RPM. The suspension thus obtained continues to be stirred, whilebeing pumped by a screw pump through a filter press set at 1 atmosphere.Once this filtration is complete, it is pumped through a filter ofnormal-hexane, this being a solvent with the double action of "drying"the preparation and solubilizing any residue surfactant which may bepresent on the surface of the microspheres. The product is then put insuitable containers and stored at 4° C. The microspheres thus preparedare suspended in a phosphate buffer solution (0.01M) (ionic strength0.1M) at pH=7, containing a concentration of calcitonin such that aprotein titer of 15 I.U. per mg of suspended microspheres is reached.After 15 minutes of stirring with a semiautomatic system the containeris immersed in liquid nitrogen until the suspension is completelyfrozen.

Once frozen, the suspension is freeze-dried for 24 hrs and the productstored at 4° C.

The mean particle size is 30 μm. The quantity of incorporated calcitoninis 13 I.U. per mg of microspheres.

EXAMPLE 37

Microspheres were prepared containing different concentrations ofcalcitonin, mainly between 5×10⁻³ and 10 I.U. per mg of microspheres,with different hyaluronic acid derivatives. Polymers with differentdegrees of hydrophobicity were used. The following are reported asexamples:

HYAFF-11 (HA totally esterified with benzyl alcohol)

HYAFF-11 p75 (HA partially esterified with benzyl alcohol)

HYAFF-7 (HA totally esterified with ethyl alcohol)

The microspheres were prepared as reported in Examples 32-33-34-35-36,and tested in an in vivo animal model for the subcutaneous absorption ofcalcitonin. In this animal model, formulations were tested with theprotein, either incorporated internally in the polymer matrix, oradsorbed externally on the surface of the microspheres. The formulationswere known as:

Formulation 1: microspheres of HYAFF-11 containing calcitonin physicallyincorporated at a concentration of 5×10⁻³ I.U. per mg of microspheres

Formulation 2: microspheres of HYAFF-11 containing calcitonin adsorbedon their surface at a concentration of 5×10⁻³ I.U. per mg of powder.

Formulation 3: microspheres of HYAFF-11 p75 containing calcitoninphysically incorporated at a concentration of 5×10⁻³ I.U. per mg ofmicrospheres.

Formulation 4: microspheres of HYAFF-7 containing calcitonin adsorbed ontheir surface at a concentration of 5×10⁻³ I.U. per mg of powder.

Formulation 5: buffered solution containing calcitonin at aconcentration of 5×10⁻³ I.U.

The microspheres were administered to male Wistar rats (115-125 gr) bysubcutaneous route. The rats were not fed for 20 hrs before theexperiment. For each formulation, 30 mg of microspheres were suspendedin 20 ml of diluting solution, composed of a mixture of 425 ml of sodiumacetate (1%) and 31.25 ml of 16% gelatin brought to pH 4.00 with HCl 1N.Subsequently, 0.4 ml (3 m I.U.) of each formulation were injectedsubcutaneously.

At fixed times of 1, 2, 3, 4 and 5 hrs after administration, 3 ml ofblood were drawn from the abdominal aorta, and the animal wassacrificed. Blood calcium was then determined directly on the serum byatomic absorption.

Table 1 shows the decrease in blood calcium values over time for all theformulations tested. From this table it is possible to see that thepolymer has a clear delaying effect on calcitonin release, which hasevident repercussions on the times and behaviour of the Ca++ decrease inthe blood.

                  TABLE 1                                                         ______________________________________                                                   Blood Ca.sup.+2 decrease (%)                                         at different times (hrs)                                                    Formulation  1        2     3      4   5                                      ______________________________________                                        HYAFF-11 (int)                                                                              5       18    30     27  13                                       HYAFF-11 (ext) 10 30 26 21 10                                                 HYAFF-11p75 (int) 10 30 28 15 3                                               HYAFF-7 (ext) 12 28 24 17 2.8                                                 Calcitonin sol. 30 22 15 10 2.5                                             ______________________________________                                    

The results also show that there is an effect due to the type ofincorporation, where in the case of microspheres containing calcitoninphysically incorporated in the polymer matrix the release is slower.

EXAMPLE 38

Microspheres were prepared which contained calcitonin at variousconcentrations, mainly 5×10⁻³ and 10 I.U. per mg of microspheres) withdifferent hyaluronic acid derivatives. Polymers with different degreesof hydrophobicity were used.

The following are examples:

HYAFF-11 (HA totally esterified with benzyl alcohol)

HYAFF-11 p75 (HA partially esterified with benzyl alcohol)

In the animal model devised to study vaginal absorption, formulationswere tested having the protein incorporated within the microspheres orexternally adsorbed on their surfaces.

The formulations were coded as follows:

HYAFF-11: microspheres containing calcitonin incorporated internally ata concentration of 5 I.U. per mg of microspheres.

HYAFF-11 p75: microspheres containing calcitonin incorporated internallyat a concentration of 5 I.U. per mg of microspheres.

Calcitonin: calcitonin dissolved in 0.9% saline brought to pH 4 withhydrochloric acid at a concentration of 100 I.U./ml.

The above formulations were administered to female, oophorectomized,Wistar rats weighing 150-200 gr. The rats were anesthetized andtracheotomized. The carotid artery and the jugular vein werecatheterized to allow blood withdrawal and replacement with saline atthe established times.

The calcitonin solution was instilled into the vagina at a dose of 400ul/kg, equal to 100 I.U./kg. The same volume of saline, pH=4, in theabsence of calcitonin, was administered to a group of control rats.

The microspheres were administered at a dose of 100 I.U. 20 mg/kg byinserting a catheter into the vagina and spraying the microspheres indry powder form.

The blood samples were gathered in heparin-treated containers and theplasma, obtained by centrifugation, was stored at -20° C. untilanalysis.

The concentration of calcium in the plasma was measured by spectroscopyin atomic absorption and expressed as a percentage of the initialconcentration in the plasma.

FIG. 1 shows the decline in blood calcium after vaginal administrationof 100 I.U. calcitonin. A decrease can be seen in blood plasma after 15minutes while the maximum hypocalcemic effect is reached after 2 or 3hours.

The small standard deviation revealed only slight animal-to-animalvariability. FIG. 2 shows calcium levels in the plasma after vaginaladministration of calcitonin formulated in HYAFF-11 and HYAFF-11 p75microspheres.

Both formulations proved effective in absorbing blood calcium,indicating that the protein had not undergone degradation in the processof preparation of the microspheres.

The difference in plasma profiles obtained with the two formulations canbe attributed to the different chemical-physical characteristics of thepolymeric matrix.

The release of calcitonin from the microspheres and consequently itsactivity is slower in the case of HYAFF-11, where the polymer is totallyesterified with benzyl alcohol, than in the case of HYAFF-11 p75. Inthis case the polypeptide was incorporated in a partially esterifiedmatrix which, in the presence of the biological fluids, is hydrated morerapidly than the totally esterified polymer, and consequently thecalcitonin spreads more quickly through the polymeric matrix into thevaginal mucosa.

The release of calcitonin can, therefore, be modulated according to thedegree of esterification of the polymer.

EXAMPLE 39

Microspheres containing insulin were prepared and tested in an in vivomodel.

The preparations of Na-insulin were administered through the nose tosheep, both as free insulin in solution and in association withhyaluronic acid derivatives in the form of microspheres prepared asreported in Examples 1-2-3-4-5.

To administer the solution, a 35-cm tube was inserted into the nasalcavity, taking care to place it at the set depth of 10 cm from theopening of the nostril.

For the administration of formulations in powder form, a 6.5-μmintratracheal tube was filled with the established dose of microspheresand placed in the nostril. In this case the device was placed at a depthof 6 cm from the opening of the nostril. Insulin was administered 2I.U./Kg by intranasal route both in solution and associated withmicrospheres. Groups of four sheep were used for each experiment. Thefollowing formulations were prepared:

Formulation 1: for nasal administration of insulin associated withhyaluronic acid (HYAFF-11) microspheres a freeze-dried product wasprepared (2.0 mg/kg corresponding to 2 I.U. insulin).

Formulation 2: for nasal administration of insulin associated withmicrospheres of hyaluronic acid (HYAFF-11 p75) a freeze-dried productwas prepared (2.0 mg/kg corresponding to 2 I.U. insulin).

Formulation 3: for nasal administration of insulin associated withmicrospheres of hyaluronic acid (HYAFF-7) a freeze-dried product wasprepared (2.0 mg/kg corresponding to 2 I.U. insulin).

Formulation 4: for nasal administration to controls a freeze-driedproduct was prepared of microspheres alone (HYAFF-11) (2.0 mg/kg).

Formulation 5: for nasal administration of insulin in aqueous solution asolution at a concentration of 2 I.U./ml was prepared.

At the set times, a 5-ml sample of blood was taken each previouslycannulated jugular vein. Each blood sample was divided in half, 2.5 mlwas placed in a heparin-treated test tube for insulin analysis, whilefor glycemia analysis the other 2.5 ml was placed in a test tubecontaining fluoride oxalate.

Table 2 shows the lowering of the plasma levels of glucose obtainedafter nasal administration of insulin.

    ______________________________________                                                Blood glucose decrease (%) at different times (min)                     Formulation                                                                             10    20   30  40  60  80  90   120  140  190                     ______________________________________                                            1   7     15     25  40  65  60  51   42   21   7                           2          10  20  39  58   49  30  21   7    0  0                            3          12  19  35  57   40  29  18   5   0   0                            4          0   0    0   0   0   0   0    0   0   0                            5          4   6    8   7   6   0   0    0   0   0                          ______________________________________                                    

It can be clearly seen from Table 2 that insulin administered throughthe nose as a simple solution (Formulation 5) has no significant effecton the lowering of blood glucose levels. It is possible to note adifferent profile in the lowering of blood glucose levels according tothe type of hyaluronic acid ester used to prepare the micropheres. Themicrospheres made with HYAFF-7 (the most hydrophilic derivative) have aless "active" interaction with the nasal mucosa and consequently lesseffect on the lowering of blood glucose levels (Formulation 3).

The microspheres made with HYAFF-11 p75 (Formulation HYAFF-11(Formulation 1) on the other hand, have a more marked interaction withthe mucosal cells and in this case too the decrease in glycemia iscorrelated with the type of derivative: HYAFF-11 p75 has less effectthan HYAFF-11, showing that the derivative's chemical-physicalcharacteristics can pilot the release of the protein.

EXAMPLE 40

The preparations of Na-insulin were administered through the nose tosheep, both as free insulin in solution and in association withhyaluronic acid derivatives in the form of microspheres prepared asreported in Examples 1-2-3-4-5.

To administer the solution, a 35-cm tube was inserted into the nasalcavity, taking care to place it at the set depth of 10 cm from theopening of the nostril.

For the administration of formulations in powder form, a 6.5-μmintratracheal tube was filled with the established dose of microspheresand placed in the nostril. In this case the device was placed at a depthof 6 cm from the opening of the nostril. Insulin associated withmicrospheres of HYAFF-11 was administered by intranasal route at dosesof 1-2-4-8 I.U./Kg. Groups of four sheep (about 40 kg) were used foreach experiment. The following formulations were prepared:

1) HYAFF 11 containing insulin at a concentration of 1 I.U./mg ofmicrospheres

2) HYAFF 11 containing insulin at a concentration of 2 I.U./mg ofmicrospheres

3) HYAFF-11 containing insulin at a concentration of 4 I.U./mg ofmicrospheres

4) HYAFF-11 containing insulin at a concentration of 8 I.U./mg ofmicrospheres

Each sheep in each group was treated with a dose of 2 I.U./kg of insulinassociated with different quantities of microspheres.

At the set times, a 5-ml sample of blood was taken from each previouslycannulated jugular vein. Each blood sample was divided in half, 2.5 mlwas placed in a heparin-treated test tube for insulin analysis, whilefor glycemia analysis the other 2.5 ml was placed in a test tubecontaining fluoride oxalate.

FIG. 3 shows mean values of the blood glucose concentrations afteradministration of 2.0 I.U./kg of insulin associated with 2.0-1.0-0.5 or0.25 mg/kg of HYAFF-11.

These results show that all the formulations cause a marked decrease inglycemia and that the profile of this decrease was similar for eachnasal administration.

FIG. 4 shows insulin levels in plasma obtained after nasaladministration of the aforesaid formulations. The graph shows theappearance of a plasma insulin peak for all formulations between thefirst fifteen and thirty minutes after administration. The plasmainsulin profile is similar for all formulations. Analysis of the areaunder the curve of the plasma insulin concentration shows that the onlystatistically significant difference is to be found between the highestdose and the lowest dose of HYAFF-11. It can also be seen that aneightfold increase in the dose of HYAFF-11 only doubles the area underthe curve. In conclusion, the results show that this system can beapplied to humans where low doses of microspheres can be used toadminister insulin.

EXAMPLE 41

Microspheres of different dimensions of HYAFF-11 containing GM1 (10-40μm) were prepared as described in Examples 29-30.

The ganglioside was administered by intramuscular route to groups of NewZealand rabbits (weighing 2.5 kg) at a concentration of 2 mg/kg.

At the set times, 2.5 ml of blood was withdrawn from the median arteryof the ear. Each blood sample was incubated at 37° C. for one hour andthen refrigerated overnight at 4° C. The samples were then centrifugedand the upper layer of serum was removed. 2 ml of tetrahydrofurane (THF)were added to 500 μl of serum for the determination of the GM1 content.The sample was stirred and centrifuged and the upper layer was stored.The precipitate was treated twice with 2 μl of THF and 500 μl ofphosphate buffer (50 mM) and centrifuged. The upper layers thus obtainedwere extracted with ethyl ether after separation of the aqueous phase,the organic phase was further extracted with water. The two aqueousphases were then mixed and freeze-dried. The freeze-dried product wasredissolved in water and the GM₁ content determined by the ELISA test.

The graph in FIG. 5 shows the levels of GM₁ in plasma afterintramuscular administration.

It can be seen that when the drug is associated with the microspheres,the time needed to reach maximum concentration in the plasma is longerthan that required by GM₁ administered on its own. Moreover, whileplasma levels of GM₁ fall rapidly when it is administered on its own,the GM₁ lasts longer in plasma when administered with microspheres,because the drug release is slower.

As reported in Table 3, which shows the areas under the curves after GM₁administration in the three different formulations. As can be seen,microspheres with larger diameters (40 μm) guarantee the presence of thedrug in the circulation for longer than those with a smaller diameter,probably because the diffusion time of GM₁ from the larger microspheresis longer.

                  TABLE 2                                                         ______________________________________                                        Area under curvce after i.m. administration of                                  GM.sub.1 at a concentration of 2 mg/kg (μg/hr/ml)                                                 HYAFF-11                                                                              HYAFF-11                                       Experiment GM.sub.1 (10 μm) (40 μm)                                   ______________________________________                                        1st study 127.74     167.06    253.4                                            2nd study 134.43 158.25 223.29                                              ______________________________________                                    

EXAMPLE 42

Microspheres containing NGF and NGF+GM₁ were prepared with differentpolymeric matrices, chosen according to the different chemical-physicalcharacteristics of the polymer (Examples 6-8-9-20-21). Three types ofthe hyaluronic acid derivatives used are reported here as examples:(HYAFF-11; HYAFF-11 p75; HYAFF-7).

To highlight the effect of the different chemical-physicalcharacteristics of the derivatives used in the preparation of themicrospheres, the release times of the incorporated products (NGF andGM1) were assessed in vitro. Samples of microspheres were suspended inphosphate buffer 0.1M (pH 7.4) u=0.3 M and kept at 37° C. under constantstirring. Samples were taken at various times and the fractions thusgathered were analysed by HPLC to measure the GM1 present and by anELISA method to measure NGF. The release of NGF followed a particularpattern in relation to the chemical-physical characteristics of thepolymer used to prepare the microspheres and depending on the presenceor absence of GM1 in the formulation. In those without GM1, as expected,NGF was released and quantified according to the hydrophiliccharacteristics of the matrix used. FIG. 6 reports as an example therelease of NGF from HYAFF-11 p75 in the presence/absence of GM1. It canbe seen that the release of NGF is more abundant and faster in thepresence of GM1 than in the samples without ganglioside, for this typeof microsphere.

In the case of formulations made with totally esterified hyaluronic acidderivatives, it was seen that NGF was released from the polymer matrixin low quantities, showing that there is a strong polymer-polypeptideinteraction. But when GM1 was present in the same formulations, it waspossible to observe significantly higher levels of NGF in solution(FIGS. 7-8) even though the maximum amount of NGF released was 40% ofthat incorporated, showing again the strong interaction with thepolymer. The invention being thus described, it is clear that thesemethods can be modified in various ways. Such modifications are not tobe considered divergences from the spirit and purpose of the invention,and any modification which would appear evident to an expert in thefield come within the scope of the following claims:

We claim:
 1. A method for administering a biologically active moleculeacross the nasal mucosal membrane which comprises administering aneffective amount of a smooth surface microsphere which comprises abiologically active molecule and an ester of hyaluronic acid,whereinsaid biologically active molecule is adhered to said ester of hyaluronicacid, wherein said smooth surface microsphere has a diameter of between1 μm to 100 μm, and wherein said smooth surface microsphere is formed bya process comprising:dissolving said ester of hyaluronic acid and saidbiologically active molecule in dimethylsulfoxide to produce a mixture;adding said mixture to mineral oil with stirring to produce an emulsion;adding ethyl acetate to said emulsion to produce a suspension; filteringsaid suspension; and recovering smooth surface microspheres thus formed,to said nasal mucosal membrane.
 2. The method according to claim 1,wherein said biologically active molecule is insulin.
 3. The methodaccording to claim 1, wherein the size of said microsphere is from 1 to15 microns.
 4. A method for administering a biologically active moleculeacross the nasal mucosal membrane which comprises administering aneffective amount of a microsphere which comprises a biologically activemolecule and an ester of hyaluronic acid or mixtures of said esters ofhyaluronic acids, and wherein said biologically active molecule issurrounded by or adhered to said ester of hyaluronic acid or mixturesthereof, and wherein said microsphere has a diameter of between 1 micronto 100 microns, to said nasal mucosal membrane.
 5. The method accordingto claim 4, wherein the size of said microsphere is from 1 to 15microns.
 6. The method according to claim 4, wherein said biologicallyactive molecule is insulin.